Persistance of M.tuberculosis
Molecular Basis Investigation of M.tuberculosis Persistance
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
- MED-DID/Diagnostics & Devices/Medicine
8 Project completed
Senior Project Manager
Gremyakova T A
Research Center of Molecular Diagnostics and Theraphy, Russia, Moscow
- State Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk
Project summary1. Description of the Problem
The past decade has been a time of a dramatic upsurge of tuberculosis all over the world. This fact has led to urgent intensification of TB investigations. Among tasks of priority are:
• Early diagnosis of tuberculosis
In-time identification of mycobacterial carriers makes it possible to capture the infection, thereby preventing its spread. However, bacteriological methods are low-efficient here, as bacilli of M. tuberculosis are known to take 24 hours to proliferate compared to E.coli whose period of reproduction is 20 to 30 min. Therefore, a process of bacteriological detection may take weeks. One of the most promising methods of detection is nucleic acid amplification, in particular a PCR analysis. However, clinical application of PCR has a number of restrictions to be detailed below (technical problems on PCR application in clinics and impossibility of differentiating viable and non-viable mycobacteria by using genomic DNA as object to study).
• Determination of drug resistance spectrum, and stages of the development of mycobacteria
According to clinical estimates, the resistance of M.tuberculosis to one or more anti-tuberculosis agents has been observed in nearly 50% of clinical isolates. The drug resistance is conferred by mutations in genes coding for a target protein, in genes regulating the delivery of drugs, and in those responsible for metabolism of drugs.
A number of mutations have been mapped. Mutations in the katG (catalase-coding gene), inhA, ahpC and oxyR regions appear to be responsible for isoniazid resistance. The disrupted gene rpoB (RNAase -subunit) has been found to determine resistance to rifampin, and resistance to ethambutol is due to over-expression of the gene embA or embB coding for transmembrane proteins, etc.
The success of any therapy depends mostly on knowledge of mechanisms of drug resistance. Therefore, new approaches to its rapid determination are needed.
A phase of the growth of mycobacteria or their persistence may also influence on the treatment. As mycobacteria are intracellular invaders, they tend to proliferate within macrophagal vacuoles. Mycobacteria are suggested to overcome three stages in their development:
1. Vivid reproduction
The last two stages may be considered as persistence, when mycobacteria are almost recalcitrant to any therapy.
In this context, of particular importance is to investigate mechanisms of persistence and molecular markers for getting persistence by the microorganism.
Expression of mycobacterial genes existing within macrophages would allow understanding mechanisms of on-going processes, determination of their development’s phase, and, therefore, forecast a result of therapy. Among genes, whose activated expression goes in front of persistence or simultaneously with it, are gene coding for GTP-binding protein responsible for mature phagosomes, RNAase -factors, genes oxyR and SOS-systems preventing “peroxide burst” in macrophages.
2. Advances in the field
Over ten years, the All-Russia Research Center for Molecular Diagnostics and Treatment has carried out PCR research to detect infections. Recombinant strains producing thermophilic DNA-polymerases have been produced, and a procedure for “dry” PCR procedure has been offered. There is a scientific basis allowing further DNA-based identification of microorganisms. Methods for monitoring expression of genes have been tested. They are based on determining a level of synthesized template RNAs by means of thermophilic Tthase with reversible transcriptase activity. In global practice, are widely used methods combining PCR and DNA-chips procedures to detect mycobacteria.
At the State Research Center for Applied Microbiology, microbiologists’ focus is placed on drug resistance, particularly on mapping of mutations in genes katG and rpoB conferring resistance to isoniazid and rifampin in M. tuberculosis.
3. Feasibility of the Project
To meet the goal of the Project, it is planned to design a specific PCR test kit to detect bacilli of M. tuberculosis, and determine phases of their growth. The system would comprise biogranules carrying components necessary to amplify DNA or RNA. To determine drug resistance, it is planned to test a technology of detection of point mutations responsible for rifampin resistance in a model of the gene rpoB, and to study mechanisms of isoniazid resistance by katG sequencing.
4. Uniqueness of the approach
The proposed approach to identification of drug-resistant viable mycobacterial strains is unique, both technically and scientifically, and has no analogues in the world. There were no previous attempts for molecular markers-involved determination of stages of the growth of mycobacteria.
5. Intermediate results
a) Development of new target DNA and RNA to detect TB mycobacteria and to determine phases of their life cycle;
b) Comparative study of approaches to cross- contamination in laboratory diagnosis;
c) Design of the unique technology for producing biogranules containing PCR-necessary ingredients;
d) Data on the spread of drug-resistant mycobacteria in Russia: their genetic characterization.
6. Final results
· Short-life RNAs will be screened to get appropriate targets to assess viability of mycobacteria and to determine stages of their development;
· Technical documentation for producing biogranules for neutral polymer PCR will be offered;
· Genetic data on resistance to isoniazid and rifampin in M. tuberculosis in Russia will be provided.
7. Application of results
Expected diagnostic kits may be used for studying epidemiological status of communities at risk, early diagnosis of tuberculosis and for choosing appropriate therapy based on data on drug resistance of mycobacteria and their life cycle.
8. Post-project possible cooperation
Further possible co-operation is commercialization of test-systems.